Pathogenic organisms, and also cell-damaging substances, first must adhere to the surface of the target cell in order to be able to cause infection or damage the attacked cell. This adhesion is mediated, for example, by a ligand-receptor relationship, whereby glycostructures play an important role. If these glycostructures are blocked at the target cell surface or at the ligand, an infection can be prevented.
Glycostructures also play an important role in the formation of tumors and metastases (Liotta et al., Annu. Rev. Cell Biol., 55 (1986), 1037-1057). The formation of tumors includes cellular interactions mediated by cell surface components, in particular carbohydrate-binding proteins. This mediates the adhesion of tumor cells by way of cellular adhesion molecules. Many stages in the formation of metastases also include cell-cell interactions or interactions between cells and the extracellular matrix (ECM) which are mediated by cell surface components. The extracellular matrix (ECM) consists mainly of laminin, fibronectin, and proteoglycanes, of which very many are glycosylated, and whose oligosaccharide side chains provide detection determinants for cellular adhesion molecules. Laminin is an N-bound glycoprotein with poly-N-acetyl lactosamine sequences. Metastatic spread occurs when circulating agglomerates of tumor cells, thrombocytes, and lymphocytes make contact in capillaries by way of adhesion molecules with the endothelium. This contact provides the signal for opening the endothelial cell functions. As a result, the tumor cells are able to bind to receptors on the basal membrane by way of additional adhesion molecules. After destroying the basal membrane, the tumor cells get direct access to the stroma, whereby again interactions occur between the laminin and fibronectin and the respective receptors, as was the case in the primary tumor invasion.
Important representatives of the carbohydrate-binding proteins are the galactoside-binding lectins galectin-1 and galectin-3 (Raz and Lotan, Cancer Metastasis Rev. 6 (1987), 433; Gabius, Biochim. Biophys. Acta, 1071 (1991), 1). It is known that galectin-3 promotes the embolic tumor dispersal in the circulation and increases the formation of metastases. Galectin-3 is expressed on the cell surface of many tumor cells, whereby the galectin-3 expression increases with progressing tumor development. Galectin-3 is also expressed by activated macrophages and oncogenically transformed cells or metastasis cells. Galectin-3 has a high affinity for oligosaccharides, which include polylactosamines, whereby galectin-3 binds in particular to two glycoproteins that occur in the form of several cell types, for example human colon cancer cells and human breast cancer cells. Another ligand for galectin-3 is, for example, laminin. Galectin-3, which is also expressed on the surface of endothelial cells, is also involved in the adhesion of tumor cells to endothelial cells.
U.S. Pat. No. 5,834,442 describes methods for treating cancers in mammals, in particular for treating prostate cancers, where the treatment of cancers, including the inhibition of the formation of metastases, is performed by oral administration of modified pectin, preferably water-soluble, pH-modified citrus pectin. To produce pH-modified pectin, a pectin solution is depolymerized by increasing its pH value to 10.0 and then reducing the pH value to 3.0. The modified pectin has a molecular weight of approximately 1 to 15 kd. Rats that were administered modified citrus pectin in their drinking water showed a significantly reduced formation of lung metastases compared to untreated control groups. In vitro experiments demonstrated that the adhesion of galectin-3-expressing MLL endothelial cells to rat aortic endothelial cells (RAEC) was almost completely inhibited in the presence of modified citrus pectin. Other experiments studied the effect of pH-modified citrus pectin on the colonization of MLL endothelial cells. The ability of cells to grow in semi-solid medium (anchorage independence) may be used as a criterion for cell transformation and the invasive potential of cells, since cell growth in a semi-solid medium requires cell migration and colonization. It was hereby found that modified citrus pectin was able to significantly reduce both the number of MLL colonies formed as well as their size. In the process, modified citrus pectin appears to have more of a cytostatics effect than a cytotoxic effect. The effect of modified citrus pectin on cell-cell interactions and cell-matrix interactions based on carbohydrate-mediated mechanisms, especially galectin-3-mediated interactions, were also investigated. It was found that, in contrast to non-modified citrus pectin, modified citrus pectin inhibited the adhesion of B16-F1 melanoma cells to laminin. Of laminin, it is known that it acts as a ligand for soluble galectin-3.
From EP 0 716 605 B1 it is known that the adherence of pathogenic agents, such as, for example, E. coli, to cells, in particular to epithelial cells of the gastrointestinal and genitourinary tract can be substantially (i.e., up to 90%) reduced with a specially prepared carrot soup, bladder tea, coconut milk, etc. According to this document, this effect can be attributed to the pectins contained in these plant products, which are essentially chains of 1,4-α-glycosidically bound galacturonides whose acid groups are esterified 20 to 80% with methanol and which, in addition to galacturonic acid, also may contain other sugar components, for example, glucose, galactose, xylose, and arabinose.
The document further describes that monogalacturonic acid shows no blocking of the adhesion, while a blocking of up top 91.7% or 84.6% can be found with digalacturonide and trigalacturonide respectively. This document clearly determines that the monomer galacturonic acid does not block the adhesion, and the desired blocking effect decreases along with an increasing molecular weight of the galacturonides. This means that the degree of polymerization of the desired galacturonides is DP 2 or 3. It is also required that the degree of esterification is <2%. The pectin hydrolysis products produced according to the method described in this document contain only very small portions of the di- and trigalacturonides designated as effective, however (approximately 12% related to the raw material). This production method wastes resources and causes environmental problems, because large quantities of unusable secondary products are created and must be disposed of.
The technical problem underlying the present invention therefore consists of providing additional methods and means for fighting infections and for reducing and/or preventing the adhesion of harmful, in particular pathogenic, substances and organisms to eukaryotic cells, in particular mammalian cells, as well for the blocking of interactions between mammalian cells, in particular tumor cells, which are mediated by carbohydrate-binding galectin-3 molecules located on the cell surface and are responsible for the development of, in particular, tumor diseases, in particular for the prevention of the formation of metastases in mammals.
This technical problem is solved with the methods according to the invention for the production of pectin hydrolysis products, which result in the production of oligogalacturonides with a monomer content as low as possible, a high content of molecules with at least one double bond each, as well as a degree of esterification of ≧20%, and which can be performed with a substantially higher yield than with the state of the art.
The problem is solved in particular in that an aqueous solution or suspension of a pectin or pectin-containing, in particular plant, material, preferably a pectin with a high degree of esterification, is treated in a first step with a first pectin-hydrolyzing enzyme A and then in a second step with a second pectin-hydrolyzing enzyme B. This yields a previously defined pectin hydrolysis product that has excellent properties as a means for reducing or preventing the adhesion for the life and/or proliferation functions of cells of harmful, for example pathogenic, allergenic, infectious, or toxic substances or organisms, for example microorganisms, such as yeasts, fungi, germs, bacteria, viruses, spores, viroids, prions.
Enzyme A may be, for example, a pectinlyase (EC 4.2.2.10) or endopolygalacturonase (EC 3.2.1.15), preferably a pectinlyase, however. Enzyme B may be an endopolygalacturonase or a pectinlyase, preferably an endopolygalacturonase, however.